Yeast cytosine deaminase is an attractive candidate for anticancer gene therapy because it catalyzes the deamination of the prodrug 5-fluorocytosine to form 5-fluorouracil. We report here the crystal structure of the enzyme in complex with the inhibitor 2-hydroxypyrimidine at 1.6-Å resolution. The protein forms a tightly packed dimer with an extensive interface of 1450 Å 2 per monomer. The inhibitor was converted into a hydrated adduct as a transition-state analog. The essential zinc ion is ligated by the 4-hydroxyl group of the inhibitor together with His 62 , Cys 91 , and Cys 94 from the protein. The enzyme shares similar active-site architecture to cytidine deaminases and an unusually high structural homology to 5-aminoimidazole-4-carboxamide-ribonucleotide transformylase and thereby may define a new superfamily. The unique C-terminal tail is involved in substrate specificity and also functions as a gate controlling access to the active site. The complex structure reveals a closed conformation, suggesting that substrate binding seals the active-site entrance so that the catalytic groups are sequestered from solvent. A comparison of the crystal structures of the bacterial and fungal cytosine deaminases provides an elegant example of convergent evolution, where starting from unrelated ancestral proteins, the same metal-assisted deamination is achieved through opposite chiral intermediates within distinctly different active sites.Cytosine deaminase (CD, 1 EC 3.5.4.1) catalyzes the deamination of cytosine to uracil and 5-methylcytosine to thymine. The enzyme has been found in bacteria and fungi, where it plays an important role in pyrimidine salvage. However, it is not present in mammalian cells, which utilize cytidine deaminase (CDA) instead (1). The bacterial and fungal CDs are distinct from each other and have evolved separately. The 426-residue hexameric Escherichia coli enzyme like the murine adenosine deaminase belongs to the (/␣) 8 -barrel amidohydrolase superfamily, in which four histidines and one aspartate located at similar spatial positions are conserved for metal coordination and enzyme catalysis (2-4). On the other hand, the 158-residue dimeric yeast counterpart may share two conserved signature sequences, HXE and CXXC, with a variety of deaminases, and thus has been grouped into the cytidine and deoxycytidylate deaminase family in the Pfam protein family data base (5, 6). The crystal structure of E. coli CDA reveals that the signature sequences contain a zinc binding motif, with histidine and two cysteines acting as zinc ligands while the glutamate serves as a proton shuttle (7).The antimetabolite 5-fluorouracil (5-FU) is one of the most active chemotherapeutic agents for the treatment of colorectal cancer, but it has limited efficacy due to gastrointestinal and hematological toxicities (8). Because of its ability to convert the relatively nontoxic 5-fluorocytosine (5-FC) into 5-FU and its absence in mammalian cells, CD has become an attractive candidate for the reduction of 5-FU toxicity toward n...
Hepatitis C virus nonstructural protein 3 (HCV NS3) helicase is believed to be essential for viral replication and has become an attractive target for the development of antiviral drugs. A fluorescence resonant energy transfer helicase assay was established for fast screening of putative inhibitors selected from virtual screening using the program DOCK. Soluble blue HT (1) was first identified as a novel HCV helicase inhibitor. Crystal structure of the NS3 helicase in complex with soluble blue HT shows that the inhibitor bears a significantly higher binding affinity mainly through a 4-sulfonatophenylaminophenyl group, and this is consistent with the activity assay. Subsequently, fragment-based searches were utilized to identify triphenylmethane derivatives for more potent inhibitors. Lead optimization resulted in a 3-bromo-4-hydroxyl substituted derivative 12 with an EC(50) value of 2.72 microM to Ava.5/Huh-7 cells and a lower cytotoxicity to parental Huh-7 cells (CC(50) = 10.5 microM), and it indeed suppressed HCV replication in the HCV replicon cells. Therefore, these inhibitors with structural novelty may serve as a useful scaffold for the discovery of new HCV NS3 helicase inhibitors.
BackgroundEstradiol plays an important role in the regulation of collagen metabolism. Deficiency of estradiol has been reported to be associated with the degeneration of many connective tissues. However, the association of estradiol and hypertrophy of the ligamentum flavum was seldom explored. Therefore, we studied the effects of estradiol on cultured cells from the ligamentum flavum.MethodsPrimary cultures of human ligamentum flavum cells obtained from surgical specimens of 14 patients undergoing spinal surgery were used to investigate the effect of estradiol on cell proliferation and the expression of collagen, elastin, and matrix metalloproteinases. Downstream pathways of estrogen receptor underlying the regulation of metalloproteinases were also investigated.ResultsIn our study, we revealed the existence of estrogen receptors on both female and male ligamentum flavum cells with a gender difference. 17β-estradiol increased early (24 hours) proliferation of ligamentum flavum cells in a dose dependent manner and the effect could not be seen when the cell density increased. Estradiol with a concentration of 10-9 M decreased collagen levels and increased the expression of MMP-13. Adding an antagonist of PI3K downstream pathway could reverse the expression of MMP-13 caused by estradiol.ConclusionsThe results implied estradiol regulated the expression of MMP-13 via PI3K pathway and contributed to the homeostasis of extracellular matrix in the ligamentum flavum.
Autosomal dominant hypocalcified amelogenesis imperfecta (ADHCAI; OMIM #130900) is a genetic disorder exhibiting severe hardness defects and reduced fracture toughness of dental enamel. While the condition is nonsyndromic, it can be associated with other craniofacial anomalies, such as malocclusions and delayed or failed tooth eruption. Truncation mutations in FAM83H (OMIM *611927) are hitherto the sole cause of ADHCAI. With human genetic studies, Fam83h knockout and mutation–knock-in mouse models indicated that FAM83H does not serve a critical physiologic function during enamel formation and suggested a neomorphic mutation mechanism causing ADHCAI. The function of FAM83H remains obscure. FAM83H has been shown to interact with various isoforms of casein kinase 1 (CK1) and keratins and to mediate organization of keratin cytoskeletons and desmosomes. By considering FAM83H a scaffold protein to anchor CK1s, further molecular characterization of the protein could gain insight into its functions. In this study, we characterized 9 kindreds with ADHCAI and identified 3 novel FAM83H truncation mutations: p.His437*, p.Gln459*, and p.Glu610*. Some affected individuals exhibited hypoplastic phenotypes, in addition to the characteristic hypocalcification enamel defects, which have never been well documented. Failed eruption of canines or second molars in affected persons was observed in 4 of the families. The p.Glu610* mutation was located in a gap area (amino acids 470 to 625) within the zone of previously reported pathogenic variants (amino acids 287 to 694). In vitro pull-down studies with overexpressed FAM83H proteins in HEK293 cells demonstrated an interaction between FAM83H and SEC16A, a protein component of the COP II complex at endoplasmic reticulum exit sites. The interaction was mediated by the middle part (amino acids 287 to 657) of mouse FAM83H protein. Results of this study significantly extended the phenotypic and genotypic spectrums of FAM83H-associated ADHCAI and suggested a role for FAM83H in endoplasmic reticulum–to–Golgi vesicle trafficking and protein secretion (dbGaP phs001491.v1.p1).
patent ducts. Consequently, surgery still remains the criterion standard of treatment for paravalvular leaks while the technology of interventional closure is being advanced.Our program comprised 1842 heart valve surgeries in 2006, including 26 patients (0.14%) operated on for para-valvular leaks. Even in this large series, the 3 cases presented here are unusual because of the multiple previous surgeries. These cases were technically challenging and of high risk, and avoiding fourth-time cardiac surgery for reoperative valve exchange by use of catheter-based intervention would have been preferable. We therefore advocate a multidisciplinary approach that includes interventional cardiologists and surgeons. Depending on level of hemolysis, severity of regurgitation, and anatomy of the paravalvular leak, the risk of surgery must be weighed against the likelihood of success of intervention. Both treatment options are clearly explained to the patient so that an informed decision can be made. Surgical backup in case of interventional complications is provided for every intervention as well. Future studies, perhaps with more suitable device technology, may address a hybrid approach, so that outcomes can be improved even for most complex cases of paravalvular leakage.
The specific CBCT acquisition, reconstruction, and CT number modification can generate accurate dose calculation for the potential use in adaptive radiotherapy.
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